This invention relates to a recombinant Rhabdovirus that expresses at least a fusion protein which facilitates fusion and entry of the recombinant Rhabdovirus into a cell target. This invention includes a recombinant Vesicular Stomatitis Virus (VSV) which expresses a fusion protein on the surface of the VSV particle. These recombinant Rhabdoviruses which express a fusion protein can be used to study function and specificity of proteins not naturally found on the Rhabdovirus being engineered, and as a method of targeting abnormal and diseased cells (e.g., virus infected cells or cancer cells) for diagnostic and therapeutic purposes. This invention also discloses methods of producing recombinant Rhabdoviruses.
A. Using Viruses to Target Cells
Viruses have been engineered in the last decade to target cells, mainly for purposes of gene therapy. Gene therapy involves the delivery of a gene, often a diseased cell, and usually involves insertion of the gene into the genome of the host cell. Viruses from the families of Adenoviridae, Parvoviridae and Retroviridae have successfully been engineered not only to insert genes to cell genomes, but also to deliver the gene to specific cells or tissues.
To deliver viruses to specific cells, the virus must be able to infect that cell type. Viruses cannot typically infect all cells or even all organisms. The ability of a virus to infect a cell is based on the xe2x80x9ctropismxe2x80x9d the virus has for the host organism and the cells of that organism. For a virus to be able to infect a cell, the cell must have a receptor for a virus protein which allows the virus to recognize and bind to the cellular receptor, whereupon it enters the cell either via endocytosis, phagocytosis or macropinocytosis. Upon entry into the cell, the virus begins replicating. To infect cells for which the virus does not have a tissue tropism, the virus must be engineered to recognize and bind to a receptor on the cell or tissue of interest. Even then, a virus may still not be able to replicate, as it may require additional cellular factors not produced in that cell.
Gene therapy viral vectors typically do not kill or lyse the cells they target. Viral vectors used for gene therapy are engineered to deliver therapeutically effective DNAs with relative safety, like a drug (see for example, D. T. Curiel et al., U.S. Pat. No. 5,547,932). Some of these vectors are capable of replicating upon infection, but only in the targeted cells (F. McCormick, U.S. Pat. No. 5,677,178). Other gene therapy vectors are engineered such that they are unable to replicate. Non-replicating gene therapy vectors are usually produced using helper plasmids (see for example, G. Natsoulis, U.S. Pat. No. 5,622,856; M. Mamounas, U.S. Pat. No. 5,646,034) or packaging cells that confer genetic elements missing in the virus genome.
Gene therapy vectors have also encountered problems with overcoming the wild-type tropisms natural to the viral vector being utilized. Pseudotype viruses were created to overcome this by engineering a virus genome to contain the DNA encoding an envelope protein from another virus, even from a different virus family or genus, that would be capable of infecting the tissue or cell target. In recent years, many gene therapy patents have been issued describing adenovirus vectors (M. Cotten et al., U.S. Pat. No. 5,693,509); adeno-associated virus vectors (J. S. Lebkowski et al., U.S. Pat. No. 5,589,377); retrovirus vectors (B. O. Palsson et al., U.S. Pat. No. 5,616,487); vectors containing chimeric fusion glycoproteins (S. Kayman et al., U.S. Pat. No. 5,643,756); vectors that contain an antibody to a virus coat protein (Cotten et al.); viruses have been engineered to allow study of human immunodeficiency type 1 (HIV-1) in monkeys, a species that normally cannot be infected by HIV-1, by creating hybrid viruses (J. Sodroski et al., U.S. Pat. No. 5,654,195); and pseudotype retrovirus vectors which contain the G protein of Vesicular Stomatitis Virus (VSV) (J. C. Burns et al., U.S. Pat. Nos. 5,512,421 and 5,670,354). Some of these gene therapy vectors use methods which attempt to overcome some aspects of the tropism related problems encountered, while maintaining the efficacy of the vector for use in gene therapy.
Virus delivery vehicles have also been created for transient gene therapy, wherein expression of the gene delivered to the cell is transient and not permanent (I. H. Maxwell et al., U.S. Pat. No. 5,585,254). Vectors have been created that selectively express certain toxin-encoding genes, such as the gene for diphtheria toxin (U.S. Pat. No. 5,585,254.). Viral vectors also can be engineered to make the host cells they infect more immunogenic (U.S. Pat. No. 5,580,564).
B. Using Rhabdoviruses to Target Cells
Both Vesicular Stomatitis Virus (VSV) and Rabies Virus (RV) are members of the Rhabdoviridae family. VSV belongs to the Vesiculovirus genus, while RV belongs to the Lyssavirus genus. All members of the Rhabdovirus family possess lipid membrane envelopes which comprise the surface of the Rhabdovirus virion.
(1) Rabies Virus
A recent paper by T. Mebatsion et al. described the engineering of a Rabies Virus (RV) such that either the entire G protein was deleted or only a small portion of the G protein was expressed. This recombinant RV was further engineered such that either CD4 or CD4 and the CXCR4 co-receptor also were expressed on the envelope of the recombinant RV virion (T. Mebatsion et al., (1997) Cell 90: 941-951). Characterization of and experiments with these engineered RV pseudotype viruses demonstrated that a CD4/CXCR4 construct, which also contained the tail of the G protein, could infect cells expressing the HIV-1 envelope protein, gp120. A drawback of this viral system was that effective incorporation of the non-RV proteins (e.g., CD4 and CXCR4) only occurred when at least the tail of the G protein (a 44 amino acid cytoplasmic domain) was expressed on the virion in the form of a chimera fused to either CD4 (RV-CD4) or CXCR4 (RV-CXCR4). A recombinant RV expressing only the RV-CD4 and the truncated G protein chimera cDNA did not contain detectable amounts of CD4 in the virion. However, a recombinant RV expressing both RV-CD4 and RV-CXCR4 yielded a virus particle with both the CD4 and CXCR4 proteins in the virus envelope (T. Mebatsion et al., 1997). The authors"" conclusion was that CD4-derived proteins are incorporated only in the form of a complex with a heterologous xe2x80x9ccarrierxe2x80x9d protein. The carrier protein in the RV construct is the CXCR4 co-receptor.
(2) Vesicular Stomatitis Virus
CD4 also has been expressed in VSV particles along with all five VSV gene products: N, P, M, G and L (Schnell et al., (1996) Proc. Nat""l Acad. Sci. USA 93: 11359-11365). A more recent publication by Schnell et al., demonstrated that both CD4 and a co-receptor protein, such as CXCR4, can be expressed in virus particles even if the entire gene encoding the G protein is deleted (xcex94G) (Schnell et al., (1997) Cell 90: 849-857). This CD4/CXCR4 recombinant was produced by utilizing a complementing plasmid containing DNA encoding the G protein. The gene encoding CXCR4 was then placed downstream of the gene encoding CD4 in the xcex94G recombinant VSV. Levels of the xcex94G-CD4 virus were 25% of the levels reported for the CD4 construct which contained the G protein (Schnell et al., 1997). However, CD4 was incorporated in the recombinant virion with the same efficiency as other VSV proteins despite the absence of a G protein. When comparing the ability of the xcex94G-CD4 construct to infect HIV-1 infected Jurkat cells to the VSV xcex94G construct containing both CD4 and CXCR4 (xcex94G-CD4/CXCR4), the xcex94G-CD4 construct infected the cells at 10% the rate of the xcex94G-CD4/CXCR4 VSV recombinant. Moreover, the xcex94G-CD4/CXCR4 was able to reduce the number of HIV-1 positive cells. These constructs were demonstrated to be capable of entering and propagating in cells infected with HIV-1 or that express the HIV-1 envelope protein (Schnell et al., 1997).
Although VSV and RV are members of the same virus family, Rhabdoviridae, VSV can produce an infectious virus particle in the absence of any G protein. In contrast, the RV recombinants could only function if the non-RV proteins were presented as a chimera containing the G tail fused to the non-VSV protein (Mebatsion et al., 1997). The ability to express a non-VSV protein in the lipid envelope of the VSV virion by itself and not as a chimera allows for the greater likelihood that the non-VSV protein, such as CD4, will form the same three-dimensional conformation that is found on the cell surface.
(3) Recombinant VSV with a Non-VSV Coreceptor Protein
A drawback of using the recombinant Rhabdoviruses as described by Mebatsion et al. (1997) and Schnell et al. (1997) is that in both cases they require a co-receptor protein, such as CXCR4 (derived from human T lymphocytes) for efficient infection into HIV-1 infected cells. The Mebatsion et al. (1997) recombinant RV virus additionally requires the tail of the G protein.
(4) Viral Envelope Fusion Proteins
The penetration of an enveloped virus into a cell occurs as a consequence of fusion of the viral envelope with the plasma membrane or with an intracellular compartment such as the endosome or lysosome following endocytosis. The fusion (F) protein of the paramyxovirus simian virus 5 (SV5) mediates a fusion between the viral envelope and the cell membrane. It has been cloned and expressed in recombinant cells. Other virus envelope proteins, including the G protein of VSV, require acid pH for fusion activity in vitro, and, in contrast, the Paramyxovirus SV5 F protein is capable of causing cell fusion at neutral pH. The wild-type virus fuses to its target cell through the combined action of the F protein and the associated HN protein of paramyxovirus. Cells that are transformed to express both the F protein and the HN protein can fuse with adjacent cells to form syncytia. However, vesicles created in vitro and comprising the F protein will not fuse with a target cell unless either the viral HN protein is also present or some other antireceptor molecule such as a lectin (i.e., wheat germ agglutinin) is present. R. G. Paterson et al., (1985) Proc. Nat""l Acad. Sci. USA 82: 7520-7524.
The present invention relates to recombinant or genetically engineered Rhabdoviruses that express a heterologous xe2x80x9cF Proteinxe2x80x9d (as defined herein) to facilitate fusion of the lipid envelope of the recombinant virus to the cell membrane of a target cell. Such constructs overcome the limitations of systems known in the art that require specific co-receptors or exhibit specific target cell tropisms.
This invention relates to a recombinant Rhabdovirus comprising at least a heterologous F Protein or polypeptide fragment thereof that is effective to facilitate the fusion of the recombinant virus to a target cell membrane. A preferred embodiment of the invention uses the F protein of Paramyxovirus strain SV5 or a polypeptide fragment thereof as the F Protein as defined herein. The preferred recombinant Rhabdovirus can be Vesicular Stomatitis Virus (VSV) or Rabies Virus (RV). In the instance of Rabies Virus, the DNA encoding the Fusion Protein is preferably fused to a cDNA encoding a portion of the RV G protein, particularly the xe2x80x9ctailxe2x80x9d portion thereof.
The recombinant Rhabdoviruses contemplated by this invention may further express a second heterologous (i.e, another non-Rhabdovirus, non-RV or non-VSV protein). This heterologous protein may be used as an attachment protein or antireceptor to target the recombinant virus to a particular receptor present on the cell membrane of the target cell. Such heterologous attachment proteins preferably recognize and bind specifically to glycoproteins or proteins that are expressed on diseased or abnormal cells as part of the disease process. A preferred attachment protein is the CD4 protein, or derivatives thereof, that function to target a recombinant Rhabdovirus to cells infected with HIV that express GP120 proteins on their cell membranes. Other receptor proteins expressed on diseased or abnormal cells, to which corresponding attachment proteins may be expressed by an engineered Rhabdovirus according to the present invention, may result from conditions relating to a parasitic infection, a viral infection, a bacterial infection, neoplasia, pre-neoplasia, leukoplakia, polyps, dermatological conditions (e.g., cafxc3xa9 au lait spots) and benign tumors.
The present invention further relates to a method of producing a recombinant Rhabdovirus which expresses an F Protein or polypeptide fragment thereof effective to facilitate fusion of the Rhabdovirus to a cell membrane. The method includes the steps of: (A) inserting a cDNA encoding Rhabdovirus N, P, L and G proteins into a suitable cell; (B) inserting a polycistronic cDNA copy of the Rhabdovirus genome containing at least the 3xe2x80x2 and 5xe2x80x2 Rhabdovirus leader and trailer regions containing the cis acting signals for Rhabdovirus replication, the genes encoding the N, P, M, and L Rhabdovirus proteins and a gene encoding an F Protein or polypeptide fragment thereof into the suitable cell; (C) culturing the cell under conditions that permit production of the recombinant Rhabdovirus; and (D) isolating said recombinant Rhabdovirus.
The foregoing method may further comprise a means of expressing another non-Rhabdovirus protein or polypeptide fragment thereof. This additional heterologous protein will usually serve as an attachment protein and therefore should have the ability to recognize and bind to a receptor expressed on the cell membrane to which the engineered Rhabdovirus is to be targeted. One preferred embodiment of this method would be to express the F protein of paramyxovirus SV5 strain as the Fusion Protein of the engineered virus
Another aspect of the present invention relates to a method of producing a recombinant Rhabdovirus which expresses an F Protein or a polypeptide fragment thereof effective to facilitate fusion of the Rhabdovirus to a cell membrane. This method includes the steps of: (A) inserting into suitable cells a polycistronic cDNA comprising at least the 3xe2x80x2 and 5xe2x80x2 Rhabdovirus leader and trailer regions containing the cis acting signals for Rhabdovirus replication, the genes encoding the Rhabdovirus N, P, and L proteins and a gene encoding an F Protein or polypeptide fragment thereof; (B) infecting the cells with a minivirus comprising the cis acting signals for Rhabdovirus replication and genes encoding at least the Rhabdovirus G and M proteins; (C) culturing the cells under conditions to permit expression of the cDNA to produce the recombinant Rhabdovirus; and (D) isolating said recombinant Rhabdovirus.
Preferred recombinant Rhabdoviruses would be VSV and RV.
The method of targeting diseased cells either in vivo or in vitro with a recombinant Rhabdovirus expressing a fusion protein comprises the steps of contacting the target diseased or abnormal cell with the recombinant Rhabdovirus under conditions that would permit infection by the recombinant virus. This embodiment may require the additional expression of another non-Rhabdovirus protein on the surface of the virus particle to serve as an attachment protein or antireceptor. Said cell targeting by a recombinant Rhabdovirus may further consist of expression of a reporter protein or fluorescent protein upon infection of the targeted cell. The targeted cell may additionally be diseased or infected. One recombinant Rhabdovirus contemplated would be one which expresses the F protein of Paramyxovirus SV5 as mediating the step of fusion.
The recombinant Rhabdoviruses described above are further contemplated for treating a subject suffering from a viral, parasitic or bacterial infection comprising administering to a patient a therapeutically effective amount of the recombinant Rhabdovirus that expresses the fusion protein. This recombinant Rhabdovirus ideally would be able to recognize and differentiate the cells infected with a virus, bacteria or parasite from those that are uninfected. It would bind to a protein expressed on the cells which arises as a result of the infection. The non-Rhabdovirus (attachment) protein responsible for binding to the infected cells is operatively linked to the regulatory sequences of the recombinant Rhabdovirus genes. Said method is similarly contemplated for treating a subject suffering from a disease, wherein the non-Rhabdovirus protein expressed by the recombinant Rhabdovirus expressing a fusion protein is capable of recognizing and binding to the protein expressed on the surface of the diseased or abnormal cell. The diseased or abnormal cell or tissue contemplated for treatment includes neoplastic cells, pre-neoplastic cells, benign tumors, polyps, cafe au lait spots, leukoplakias, other skin moles or lesions or dermatologic conditions.
The invention also relates to a method of identifying Fusion Proteins as defined herein. Steps of this method include: (A) inserting suitable cells with a polycistronic first cDNA containing at least the 3xe2x80x2 and 5xe2x80x2 Rhabdovirus leader and trailer regions containing the cis acting signals for Rhabdovirus replication, the Rhabdovirus genes encoding the N, P, and L proteins, a gene encoding an F Protein candidate and a non-Rhabdovirus protein; (B) infecting the cells with a minivirus containing cis acting signals for Rhabdovirus replication and a second cDNA encoding a reporter protein; (C) culturing the cells under conditions to permit replication of the first and the minivirus to produce a recombinant Rhabdovirus; (D) isolating said recombinant Rhabdovirus; (E) bringing the isolated recombinant Rhabdovirus in contact with uninfected cells under conditions permitting infection by said recombinant Rhabdovirus; and (F) determining whether the reporter protein is expressed.